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Flavonoids from Almond Skins Are Bioavailable and Act Synergistically
with Vitamins C and E to Enhance Hamster and Human LDL
Resistance to Oxidation1,2
Chung-Yen Chen, Paul E. Milbury, Karen Lapsley,* and Jeffrey B. Blumberg3
Antioxidants Research Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research
Center on Aging, Tufts University and *The Almond Board of California, Modesto, CA
Almonds (Prunus amygdalus Batsch) rank highest among
tree nuts, such as hazelnut, pecan, pistachio, and walnut, in
total annual crop production (8), and are a good source of
nutrients associated with heart health such as vitamin E,
monounsaturated fatty acids, PUFA, arginine, and potassium
(9). Almond skins, removed from the nut by hot water blanching during preparation of almond meat, constitute ⬃4% of the
total almond weight and are generally treated as a waste
product. However, an array of flavonoids, including catechins,
flavonols, and flavanones in their aglycone and glycoside
forms, were identified in almond skin (10,11). These compounds may contribute to the health benefits associated with
almond consumption. The bioavailability and pharmacokinetics of individual flavonoids were demonstrated in several studies (12–14). Catechins and flavonols from food were also
found to be bioavailable in rodent and human studies with the
food, although its matrix and preparation may have a significant effect on their bioavailabilities (15–20). However, no
studies have explored the bioavailability of flavonoids from
almond skins and their effect on antioxidant activity. Therefore, we examined almond skin flavonoids (ASF)4 for their in
vitro action with and without vitamins C and E on the resistance
Flavonoids are natural constituents of plant foods and have
been carefully studied in fruits and vegetables, but less attention has been paid to their presence in whole grains and tree
nuts (1,2). Flavonoids appear to possess a variety of biological
activities, including antioxidant, anti-inflammatory, and vasodilatory actions (3). Putative health benefits of flavonoids were
suggested by epidemiologic studies showing an inverse association between intake and risk of cardiovascular disease (4,5).
A similar relation was observed with tree nuts, leading to the
recent approval by the FDA of a qualified health claim for tree
nuts, including almonds, that eating 42.6 g/d (1.5 oz/d) as part
of a diet low in saturated fat and cholesterol may reduce the
risk of heart disease (6). It is worth noting here the randomized
clinical trial conducted by Jenkins et al. (7) demonstrating
that almond consumption can reduce total and LDL cholesterol and increase the resistance of LDL to oxidation.
Presented in part at Experimental Biology 03, April 2003, San Diego, CA
[Chen, C.-Y., Shen, J., Li, T., Milbury, P. & Blumberg, J. (2003) Bioavailability
and antioxidant activity of polyphenolics from almond skins in hamsters. FASEB
J. 17: A377 (abs.)].
Supported by the USDA Agricultural Research Service under Cooperative
Agreement No. 58-1950-4-401 and the Almond Board of California. The contents
of this publication do not necessarily reflect the views or policies of the USDA nor
does mention of trade names, commercial products, or organizations imply
endorsement by the U.S. government.
To whom correspondence should be addressed.
Abbreviations used: Cmax, maximal concentration; ECD, electrochemical
detection; GAE, gallic acid equivalents, Tmax, time to maximal concentration.
0022-3166/05 $8.00 © 2005 American Society for Nutritional Sciences.
Manuscript received 13 January 2005. Initial review completed 27 February 2005. Revision accepted 23 March 2005.
Downloaded from jn.nutrition.org by guest on March 10, 2014
ABSTRACT Consumption of tree nuts such as almonds has been associated with a reduced risk of coronary heart
disease. Flavonoids, found predominantly in the skin of almonds, may contribute to their putative health benefit, but
their bioactivity and bioavailability have not previously been studied. Almond skin flavonoids (ASF) were extracted
with HCl:H2O:methanol (1:19:80) and their content of catechins and flavonols identified by HPLC with electrochemical detection. ASF bioactivity was assessed in vitro by their capacity to increase the resistance of human LDL
to oxidation induced by 10 mol/L Cu2⫹. ASF from 0.18 to 1.44 mol gallic acid equivalent (GAE)/L increased the
lag time to LDL oxidation in a dose-dependent manner (P ⱕ 0.0001). Combining ASF with vitamin E or ascorbic
acid extended the lag time ⬎200% of the expected additive value (P ⱕ 0.05). The bioavailability and in vivo
antioxidant activity of 40 mol ASF were examined in BioF1B hamsters. Peak plasma concentrations of catechin,
epicatechin, and flavonols (quercetin, kaempferol, and isorhamnetin) occurred at 60, 120, and 180 min, respectively. The concentration of isorhamnetin was significantly elevated in liver at 180 min. Absorbed ASF enhanced the
ex vivo resistance of hamster LDL collected at 60 min to oxidation by 18.0% (P ⫽ 0.028), and the in vitro addition
of 5.5 mol/L vitamin E synergistically extended the lag time of the 60-min sample by 52.5% (P ⱕ 0.05). Thus, ASF
possess antioxidant capacity in vitro; they are bioavailable and act in synergy with vitamins C and E to protect LDL
against oxidation in hamsters. J. Nutr. 135: 1366 –1373, 2005.
The residue was also reconstituted with phosphate buffer (7. 3.59 mmol/L NaH2PO4.4) for the in vitro LDL oxidation assay. After centrifugation at 14. Flavonoid profile of almond skin. 10 – 80% (25– 60 min). 60. Analysis of plasma and liver flavonoids.4 mol/L NaH2PO4. This study was approved by the Animal Care and Use Committee of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University. BHT.6) and 20 L ␤-glucuronidase (98. we chose a test dose of 50 mg/kg body weight or 40 mol GAE/hamster because rodents consume 5– 6 times more food-based energy per unit of body weight than humans (31). The results of the LDL oxidation are expressed as lag time (defined as the intercept at the abscissa in the diene-time plot) (25). 4.5 g) frozen in liquid nitrogen were pulverized and extracted twice with 5 mL acetonitrile containing 0.4).59 mmol/L NaH2PO4. 4. and 300 min (n ⫽ 4.000 kU/L ␤-glucuronidase and 2400 kU/L sulfatase) at 37°C overnight. (-)-epicatechin. and potassium bromide were purchased from Fisher. Brinkmann Instrument) and the resulting slurry used for the hamster gavage. pH 3. Thus. An aliquot of plasma was stored at ⫺80°C for determination of flavonoid status with the remainder used immediately for analysis of LDL oxidation. The sample was centrifuged at 1000 ⫻ g for 15 min at 4°C and methanol evaporated with nitrogen gas. and ascorbic acid was dissolved in PBS and added to the reaction immediately before initiation of oxidation. and ␤-glucuronidase type H-2 (containing sulfatase from Helix pomatia). 180. Total almond skin phenolics were assessed via the Folin-Ciocalteu reaction according to Singleton et al.0 mL phosphate buffer (7. respectively). 90.5 g cholesterol/100 g for 2 wk before the acute administration of the ASF slurry (29). 120. and 150 mmol/L NaCl. After incubation with 10 L ␤-glucuronidase (98. Vitamin E (␣-tocopherol) was dissolved in methanol and subsequently diluted with phosphate buffer to obtain the selected concentrations.8 mg) was delivered in 1. pH 7. respectively. The following mobile phase gradient profile was used (% solvent B): 1% (0 –15 min). 4. All LDL experiments were performed in duplicate in each of 3 subjects. and in vivo antioxidant actions in hamsters. 1:19:80) over 16 h at 4°C. hamsters were randomly assigned on the basis of their body weight to 6 time point groups: 0 (baseline). were housed individually in cages with a 10:14 h light:dark cycle. the mixture was processed in the same manner as plasma samples.2 g (mean ⫾ SE). Formation of conjugated dienes was monitored by absorbance at 234 nm at 37°C over 6 h using a Shimadzu UV1601 spectrophotometer (Japan) equipped with a 6-position automated sample changer. deconjugation of flavonoid metabolites took longer in liver than in plasma samples. with a body weight of 136.79 mmol/L Na2HPO4. and 10 –1% (65– 68 min). (23). vitamin E (␣-tocopherol). Liver samples (0. and snap-frozen in liquid nitrogen for additional flavonoid analysis. including 2 pumps (model 582).0 mL via stomach gavage to hamsters anesthetized with Aerrane™ (Baxter). The quantity of individual almond flavonoids was calculated according to concentration curves constructed with purified standards. Because Ku¨hnau (30) estimated daily intake of flavonoids and other polyphenolics by a person weighing 70 kg at 14 mg/kg. dried under purified nitrogen. Due to a lower volume of ␤-glucuronidase and different buffer system. Plasma samples were collected after the whole blood was centrifuged at 1000 ⫻ g for 15 min at 4°C. sodium phosphate dibasic. ASF were extracted with 500 L acetonitrile. and from the last 3 subjects for experiments on the interaction between ASF with vitamins C and E. catechin. dried under nitrogen. isorhamnetin.ALMOND SKIN FLAVONOID BIOAVAILABILITY of human LDL to oxidation as well as their bioavailability. Blood from each hamster was collected from the orbital sinus via micro-hematocrit tubes coated with heparin and stored in microtubes containing ETDA. and reconstituted in 200 L of the aqueous HPLC mobile phase.nutrition. ASF and vitamin E were incubated with LDL at 37°C for 30 min before initiation of oxidation. Methanol was removed using a Rotavapor 134 (Buchi. BioBreeders) were used in the bioavailability experiments due to the similarity of their lipoprotein metabolism to that of humans (28). METHODS AND MATERIALS to reflect amounts that can be obtained from dietary intakes (26). All HPLC procedures were performed with ESA instruments. KBr and EDTA were removed from the sample using a PD-10 column (Amersham Pharmacia Biotech). the women were 28 – 64 y old with a mean body weight of 63 ⫾ 15 kg. autosampler (model 542).5.44 mol/L were selected 1367 . 1–10% (15–25 min). approved by the Institutional Review Board of the Tufts-New England Medical Center. After overnight food deprivation.4) for testing in the assay.5 m) with a 0. (22) and expressed as mol/L gallic acid equivalents (GAE).6 ⫻ 150 mm.0 ⫾ 1. Animals. 2. purified stan- Downloaded from jn. Detection was achieved with potentials applied from 60 to 720 mV with 60-mV increments. The powder (0.5%. LDL samples from the first 3 subjects were used to assess the dose-response relation of ASF. The residue was reconstituted with the aqueous mobile phase and the ASF characterized by HPLC with electrochemical detection (ECD) according to a slightly modified method of Milbury (21). ASF with 40 mol GAE (6. Folin Ciocalteu phenol reagent. pH 5.79 mmol/L Na2HPO4. pharmacokinetics. Briefly. Preparation of ASF for hamster gavage. Pulverized almond skin powder provided by the Almond Board of California was used for ASF extraction. LDL protein was determined using a bicinchoninic acid protein assay kit (Pierce). The supernatant was removed after centrifugation at 14. (24) using a Beckman NVT-90 rotor in a Beckman L8-mol/L centrifuge (329. ASF in hamster plasma and liver were assessed by the HPLC-ECD as described above. combined. LDL was separated from plasma according to Chung et al.0 mL saline was administered to the baseline group. Analyte separation was achieved using a Zorbax ODS C18 column (4. levels generally found in healthy people (27). To increase lipoprotein production for subsequent collection. 20 L vitamin C-EDTA (200 mg ascorbic acid plus 1 mg EDTA in 1.01% BHT. sodium phosphate monobasic.000 kU/L ␤-glucuronidase and 2400 kU/L sulfatase) were added to 200 L plasma and the mixture was incubated at 37°C for 45 min.5 g) was extracted twice with 10 mL acidified methanol solution (HCl:H2O:methanol.000 ⫻ g for 5 min. An aliquot of ASF in acidified methanol was dried under nitrogen and redissolved in an equal volume of phosphate buffer (pH 7. BioF1B strain Golden Syrian Hamsters (n ⫽ 22. The 1-y-old hamsters. food-grade ascorbic acid. ASF were extracted with acidic aqueous methanol as described above with the substitution of white vinegar for HCl. The antioxidant capacity of ASF was assessed in vitro with human LDL according to a slight modification of the method described by Esterbauer et al. Antioxidant capacity of ASF in human LDL. 500 L of supernatant was removed following a vigorous vortex for 30 s and centrifugation at 14. Concentrations ofASF from 0.18 to 1. The following reagents were obtained from Sigma Chemical: copper sulfate. pH 7. All organic solvents. quercetin. 4. rinsed with saline. The identification of individual ASF was achieved by comparing its retention time and electrochemical response with purified standards obtained from Sigma Chemical. kaempferol.6 mL/min flow rate and mobile phase gradient from mobile phase A (75 mmol/L citric acid and 25 mmol/L ammonium acetate in 90% H2O and 10% acetonitrile) to mobile phase B (75 mmol/L citric acid and 25 mmol/L ammonium acetate in 50% H2O and 50% acetonitrile) for 68 min. The effect of ASF plus vitamin C or E on the resistance of LDL to oxidation was expressed as the lag time increase compared with the lag time of LDL without the addition of ASF or vitamin C or E.0 mL of 0. and 2. sodium chloride. Concentrations of vitamin E and C employed in these experiments were 9 –18% and 3– 6% of plasma concentrations. Whole liver was removed. was performed between 1400 and 1430 h in 6 healthy Caucasian women who were not fasting. 2. Spiked. 100 L of supernatant was injected into the HPLC.org by guest on March 10.000 ⫻ g for 5 min. hamsters consumed ad libitum a nonpurified diet (Harlan) enriched with 10 g coconut oil and 0. Collection of venous blood. and Coularray 5600 A detector. 1. glacial acetic acid. the final concentration of methanol was 0. 2014 Chemicals and reagents.000 ⫻ g for 5 min. and reconstituted in 500 L of 1 mol/L sodium acetate buffer. LDL (182 nmol/L) were oxidized by 10 mol/L CuSO4 in a total volume of 1.271 ⫻ g for 90 min). and 150 mmol/L NaCl. 80 –10% (60 – 65 min).
1). (2) epicatechin. n ⫽ 3. indicating the presence of numerous potential antioxidant compounds in almond skin (Fig.5 ⫾ 0. Downloaded from jn. Lag time of control (no added ASF. rather than an internal standard.5 min. Values are means ⫾ SE. (4) kaempferol. VC or VE.5 mol/L before initiation of oxidation. A paired t test was performed to determine the significance of the synergy between ASF and vitamins C or E in the in vitro human LDL oxidation by comparing the observed lag time during co-incubation with the expected (calculated) sums of values observed for ASF and vitamin C or E treatment alone.7 min and (B) 47. . The recovery rates of spiked standards ranged from 75 to 80%. The JMP IN 4 statistical software package (SAS Institute) was used to perform all statistical analyses.0 mL phosphate buffer (pH 7. 1368 FIGURE 2 Effect of ASF on lag time to Cu2⫹-induced oxidation of human LDL in vitro. All results are reported as means ⫾ SE.CHEN ET AL. were square root-transformed before ANOVA analysis (2).3 ⫾ 3. Values are means ⫾ SE. and kaempferol in liver. Differences with P ⱕ 0.05. Ex vivo antioxidant capacity of ASF.2 mol GAE/g. LDL (182 mol/L) was oxidized by 10 mol/L Cu2⫹ with the addition of ASF. FIGURE 3 The synergistic effect of ASF and vitamin C or E (VC or VE) on the lag time of human LDL oxidation in vitro. or VE) ⫽ (A) 45. and in vitro hamster and human LDL oxidation after significant differences were obtained by one-way ANOVA. Data from all time points was included in correlation tests. Each trace reflects the electrochemical response of a specific applied potential (as mV) in the ECD. epicatechin. †P ⱕ 0. Because hamster LDL obtained through ultracentrifugation was lower than human LDL (determined by protein content). VC. The equal variance assumption was assessed by Hartley’s test (32) and data.nutrition. 2014 dards. P ⱕ 0. RESULTS The total phenolic content in the almond skins as determined by the Folin-Ciocalteu method was 8.7 min. ‡P ⱕ 0. Statistics. The TukeyKramer honestly significant difference test was used in experiments on ASF in hamster plasma. The percentage value above the solid bar indicates the observed synergy greater than the calculated sums of the individual treatments.7 ⫾ 1. quercetin. Lag time of control (no added ASF) ⫽ 49.05 were considered significant. including epicatechin and isorhamnetin in plasma and catechin. (5) isorhamnetin. Means without a common letter differ. Five aglycone flavonoids were identified and quantified as follows FIGURE 1 HPLC-ECD profile of ASF. n ⫽ 3. Labeled peaks are: (1) catechin.4). were added at the beginning of sample processing to construct standard curves and account for extraction losses and quantify concentrations (2). The difference in lag time of ex vivo hamster LDL oxidation between baseline and ASF administration was determined by a simple t test. Symbols indicate different from the calculated sums: *P ⱕ 0. Based on the synergistic results obtained with ASF plus vitamin E in the in vitro experiments. Bars are stacked to illustrate the calculated additive effect of the 2 treatments.01. (3) quercetin.005. The ex vivo resistance of hamster LDL to Cu2⫹-induced oxidation was performed as described above for the in vitro experiments to test antioxidant activity of flavonoids in ASF-gavaged hamsters. HPLC-ECD chromatography resolved ⬃30 peaks with detectable redox potential. The unit of the ordinate is nanoamp (nA). vitamin E was added in vitro to hamster LDL to a final concentration of 5. or ASF ⫹ VC or VE.0001.org by guest on March 10. 91 nmol/L LDL was oxidized by 5 mol/L CuSO4 in a total volume of 1.
75 and 5. 5).001) (Fig. n ⫽ 4. vitamin E at 2. quercetin. and 180 Downloaded from jn. (5) isorhamnetin.01). (3) quercetin.4 and 24. The lag time was 289% longer than the expected additive value with the combination of 0. and 761 nmol/L.5). the maximum concentrations (Cmax) of catechin. kaempferol. . (4) kaempferol.ALMOND SKIN FLAVONOID BIOAVAILABILITY 1369 isorhamnetin were 376.1 ⫾ 1.0 mol/L increased lag time by 12.nutrition.9). an observed value twice the calculated additive value) was observed with 0. All 5 identified flavonoids from almond skins were bioavailable in hamster plasma (Fig. Based on their pharmacokinetic profile in the plasma.7 min. In vitro. quercetin (6. 133.6 and 34 ⫾ 2. isorhamnetin (20. Labeled peaks are: (1) catechin. The time to reach the Cmax (Tmax) was 60 and 120 min for catechin and epicatechin. P ⱕ 0. Ascorbic acid at 2. A 1-fold synergy (i.0001) (Fig. 3B).3 ⫾ 0. Means in each panel without a common letter differ. (nmol/g): catechin (186. 3A). epicatechin (77.6% of the total phenolic content of almond skins.e. no synergy was observed with the 2. respectively (Fig. 222.05. respectively. 72.0 mol/L ascorbic acid (P ⱕ 0. epicatechin.18 and 0. kaempferol.9 ⫾ 1. respectively. 4). respectively. and kaempferol (3.org by guest on March 10.. epicatechin.7 min.8).5 mol/L dose of vitamin C (Fig.5 mol/L increased lag time by 19.0). 2014 FIGURE 4 HPLC-ECD chromatographs of hamster plasma samples obtained at 60 min (A) and 180 min (B) after administration of 40 mol GAE ASF and immediately after gavage with saline (baseline).5 mol/L vitamin E (P ⱕ 0. Values are means ⫾ SE.36 mol/L ASF and 5. 2). (2) epicatechin.36 mol/L of ASF in combination with 5. and isorhamnetin in the plasma of hamsters administered 40 mol GAE ASF.5 and 5. quercetin. (A) 210 mV ECD trace. (B) 70 mV ECD trace. These 5 flavonoids represent 3. and FIGURE 5 Time course of catechin. ASF increased the resistance of human LDL against Cu2⫹-induced oxidation in a dose-dependent manner (P ⱕ 0.9).
Using HPLC-ECD.9 min at the baseline. and 180 min time points.44). If a prooxidant action of quercetin occurred in our study. Hwang et al. 36. 1370 TABLE 1 Correlation coefficients among flavonoids in the plasma and liver of hamsters administered 40 mol GAE ASF1 Kaempferol Plasma Quercetin Kaempferol Catechin Liver Quercetin Kaempferol Catechin Plasma and liver Isorhamnetin Isorhamnetin Epicatechin r P-value r P-value r P-value 0.08 0. likely flavonoids or related polyphenolics.4. However.5 ⫾ 1. Isorhamnetin showed a similar pharmacokinetic pattern in both plasma and liver.e.0 ⫾ 6.028 and 0.54 ⱕ0. others found a faster plasma Tmax for catechin than quercetin when single compounds were fed to rats (16. The lag time of ex vivo hamster Cu2⫹-induced LDL oxidation was 30. Correlation coefficients between the 3 plasma flavonols ranged from 0. 120.62 — — ⱕ0. However. and quercetin and urate (42).0.0 and 24. kaempferol.CHEN ET AL.16. and p-hydroxybenzoic acid (10.19. but not quercetin. most of the supportive evidence has been based only on in vitro experiments or in vivo feeding studies with a single flavonoid aglycone or glycoside (26).8 ⫾ 0.7 min. and kaempferol in liver were not affected by acute ASF gavage. 182. Extrapolations about flavonoids from in vitro results are limited because of their relatively poor general bioavailability and extensive biotransformation in vivo (14). in contrast to the 180 min observed in plasma.5. differed from those in the plasma (Fig.5% longer lag time than that collected at baseline (P ⱕ 0. DISCUSSION The putative health benefits of flavonoids have been attributed in part to their antioxidant activity (33. vitamin C may contribute to the synergy by inhibiting the decomposition of lipid peroxides and/or preventing Cu2⫹ from binding to LDL (40).3 ⫾ 1.1 ⫾ 8. The bioavailabilities of selected single catechin and flavonol compounds were reported (14. At 300 min. 6).7. respectively.0% in the ex vivo resistance of LDL to oxidation at 60 and 120 min (P ⫽ 0. and isorhamnetin) and measured ⬃25 other redox compounds (Fig. Such interactions (14. quercetin. such as glycosides of quercetin.40. epicatechin.8. Further.66 0. such as the regeneration of oxidized malvidin 3-glucoside by catechin (39).1 ⫾ 1. The Cmax of isorhamnetin in the liver was 698 nmol/g at 180 min after administration. we quantified 5 aglycones (catechin. 2014 min for the 3 flavonols. it may have been masked by other constituents of almond skin.34 — 0. Similar to our finding in hamsters.34).41) may also partly account for the synergy between the ASF and vitamins C and E observed here (Fig. vanillic acid. The complex of ASF effectively increased the resistance of human LDL to oxidation in vitro in a dose-dependent fashion within physiologically relevant concentrations (Fig. 38. . flavanones.9 ⫾ 13. When 5. catechins.0001 — — — 0. i.25 to 10 mol/L (35–37).org by guest on March 10. and 160.005 — — — 0. Filipe et al. Almond skins were previously reported to contain 3 classes of flavonoids.11).0.05). Concentrations of quercetin and kaempferol were correlated (Table 1). 3).78 — — ⱕ0. Absorbed ASF appeared to induce a small increase of 18.66 to 0. we examined the behavior of a complex array of polyphenolics in almond skins while measuring several specific flavonoid constituents. suggesting that catechin is rapidly absorbed Downloaded from jn.8. (38) reported a prooxidant activity of low concentrations of quercetin (⬍2 mol/L) indicated by increased malondialdehyde formation during Cu2⫹-induced LDL oxidation.9 ⫾ 2. (40) suggested that polyphenolics may stabilize the LDL particle structure via an interaction with the apoprotein-B domain. Isorhamnetin pharmacokinetics paralleled those of kaempferol. Similar synergies were observed between genistein and ascorbic acid (40). the lag time of LDL oxidation was 119. Concentrations of catechin.2 0. kaempferol. The Cmax of liver quercetin and kaempferol at 73 and 130 nmol/g did not differ from baseline and their Tmax was 90 min. quercetin.19 — — 0. These results are consistent with several reports of the antioxidant activity of flavonoids such as catechin and quercetin with concentrations administered ranging from 0. isorhamnetin remained higher than baseline levels after 300 min (P ⱕ 0.01 — — 0. respectively.3 ⫾ 17. 60. except for isorhamnetin.78 although no relation was noted between the catechins (Table 1). It is possible that the potential prooxidant actions of single flavonoids do not occur in natural mixtures of plant polyphenols in which opportunities for recycling oxidized compounds may exist. 152. isorhamnetin. 160. only LDL collected at 60 min showed a synergistic increase with a 52.0001 ⱕ0.9 ⫾ 3. 90.4.44). respectively). Pharmacokinetic patterns of the flavonoids in liver. and 38. but little information is available regarding the concurrent absorption profile of mixtures.2 ⱕ0. In contrast. and flavonols (10. and naringenin as well as protocatechuic acid.43. 34.0001 — — 0. 1). 2) (26).0001 — — 1 Based on one observation in 22 hamsters.nutrition..05).5 mol/L vitamin E was added in vitro to the reaction.73 — ⱕ0. In contrast. the plasma concentrations of these flavonoids were less than half the Cmax and their concentrations (with the exception of quercetin and isorhamnetin) did not differ from the baseline value. oat phenolics and ascorbic acid (2). In addition to recycling mechanisms.31 — — ⱕ0.008.11). The epicatechin concentration was significantly elevated at 300 min.0001 — — 0.
No association was noted between the plasma concentrations of the 2 catechins. 5). quercetin-4⬘-glycoside and rutin (quercetin rutinoside) have Tmax of 0. kaempferol. quercetin. Noticeable amounts of catechin. although it is possible that these flavonoids were quickly redistributed to other tissues. the weak direct response elicited in vivo may be a result of a lower potency of glucuronidated. n ⫽ 4. and isorhamnetin in the liver of hamsters administered 40 mol GAE ASF. P ⱕ 0.g. epicatechin. flavonoids were detected in mouse and rat tissues with concentrations from 30 to 3000 ng aglycone equivalents/g (13). respectively. caution is warranted when interpreting these values due to pharmacokinetic differences between species as well to other confounding variables such as differing ingredients in the diet fed and potential competition between polyphenolics for absorption (44). and kaempferol in hamster liver may be attributed to their presence in the basal nonpurified diet fed to hamsters. possibly due to direct contribution of isorhamnetin from almond skins. Manach et al. although the vitamin E content of the nut may have contributed substantially to this result.. Moon et Downloaded from jn. or quercetin in the liver. In vivo. a very modest antioxidant effect of ASF was suggested by the small increase in the resistance of LDL in hamsters treated with the almond skin extract compared with saline. The plasma pharmacokinetics of the almond flavonols were similar with high correlation coefficients (Table 1). In addition. at 300 min may reflect an unusually slow distribution to this tissue or result from the small sample size employed at this one time point. 2014 FIGURE 6 Catechin.nutrition. Halvorsen et al. e. sulfated. kaempferol.org by guest on March 10. Although isorhamnetin is a metabolite of quercetin (45). 6). and kaempferol glycosides (10. and/or methylated metabolites of the ASF. quercetin. Values are means ⫾ SE. The half-life of flavonols in the hamsters (5 h) was shorter than that in rats (11–28 h). Baba et al. However. Although a higher dose may have produced a greater effect. the association between their plasma concentrations was low. (13) for humans. we found a shorter half-life in plasma for the catechins compared with the flavonols in hamsters.11).5 and 7 h. Consistent with the antioxidant capacity of almond skins using the LDL oxidation assay. (46) demonstrated the antioxidant potency of whole almonds in vitro with the ferric reducing antioxidant power assay. The increase in hepatic catechins. with the exception of isorhamnetin. It is not clear why ASF administration did not increase catechin. although such differences could be accounted for by differences in their glycosides (12). in humans (26). The relatively high level of isorhamnetin in liver may reflect a slow rate of clearance compared with other flavonoids or a gradual contribution from methylation of aglycone and glycone quercetin. We observed a Tmax at 180 min for the 3 almond skin flavonol aglycones after deconjugation by glucuronidase and sulfatase in hamsters (Fig. the 2 catechins and 3 flavonols were detected in liver although. Although isorhamnetin status in plasma was correlated with its concentration in liver. after ASF gavage. suggesting the potential contribution to this value from unidentified flavonol glycosides in almond skins. especially epicatechin. In contrast to a Tmax of 120 min for epicatechin in hamsters (Fig.05. their pharmacokinetic patterns were quite different from those in plasma (Fig. Indeed. it was not associated with other hepatic flavonols. 1371 . Similar to the relations suggested by Manach et al. 5). quercetin and kaempferol concentrations in the liver were correlated. (44) reported a 60-min value in rats. Means in each panel without a common letter differ. Although the number of reports is limited.ALMOND SKIN FLAVONOID BIOAVAILABILITY in the upper gastrointestinal tract and cleared from plasma within several hours. In hamsters. (14) reported that ⬎90% of catechin and quercetin in rat liver was methylated. epicatechin. such as quercetin. Similar to their relation in plasma (Table 1). glycoside moieties appear to have a substantial influence on pharmacokinetics. isorhamnetin.
K.. G. 19. & Blumberg. Wadsworth Publishing Co. A. Food Chem. J. Docket No. J. Hollman. Biol. Nutr. & Trevisan. M. R.. 1372 ACKNOWLEDGMENTS We thank Donald Smith for his contributions to establishing the hamster model and Ting Li and Jennifer O’Leary for their excellent technical assistance in the laboratory. 9. Phytochemistry 65: 2391–2399. 220: 198 –202. the ASF-vitamin E synergy appeared in LDL collected at 60 min. Because almonds represent one of the richest dietary sources of vitamin E. C. C. A. Ordovas. Williamson.. Nutr. Boelens. G. & Chen. & Wolffram. & Plumb. & Jimenez.. C. J. onions. 2. Lean. Drake. Huxley. Y. Med. & Re´me´sy. Kwak. T. (2004) Polyphenolics: food sources and bioavailability... J. (1998) Structural dependence of flavonoid interactions with Cu2⫹ ions: implications for their antioxidant properties. Nutr. (2002) Flavonoid intake and risk of chronic diseases. Texier. 38: 877– 884. Fernandes. 134: 1459 –1466.. A.. 24: 117–191.. Hider. 28. H. S. .. G.. 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